Many biochemical analyses of biological systems are performed on groups of cells based on the assumption that the cells are sufficiently similar that an ensemble average from the cell group will yield a useful result. However, this approach can obscure the underlying heterogeneity of the individual cells and masks import and differences in gene expression, protein levels, and small-molecule distributions. Bringing modern methods of analysis to new levels of sensitivity and spatial resolution is necessary if single cell biochemica analysis is to be achieved. Mass spectrometry is a key analysis technique for cell biochemistry, but there are technological barriers in sampling scale that must be overcome for it to be used to its full potential with single cells. The goal of this research is to construct and test a system or nanometer scale laser ablation sampling of single cells and tissue coupled with electrospray ionization mass spectrometry. This system uses apertureless near-field laser ablation to transfer peptides, proteins and other biomolecules to a microdroplet that is used for ultra high sensitivity electrospray ionization. The project is divided into two components (1) sampling system setup, validation, and testing with biomolecule standards, which will establish detection limit and spatia resolution benchmarks, and (2) proof of concept testing with cells and tissue, which will establish a working limit of detection and spatial resolution as well as provide a benchmark sample complexity determination that will aid in designing follow-on experiments. Nanoscale laser ablation sampling developed in this project will have applications not only in mass spectrometry but also in microfluidics. Laser ablation sampling with droplet capture will provide a new method for spatially resolved sampling into a microfluidic device. It will also allow a separation step to be added to mass spectrometry imaging that will enable the imaging of minor biomolecule components of tissue. PUBLIC HEALTH RELEVANCE: Single cell analysis is necessary because the ensemble average of cell mixtures obscures important information about human health therefore it is necessary to develop new single cell analysis techniques. Mass spectrometry is one of the most important analysis tools but technological barriers prevent sampling and imaging at the single cell level if large biomolecules such as peptides and proteins are the target analyte. The goal of this research is to develop nanometer scale sampling for mass spectrometry using near-field optics and laser ablation.